Genomic architecture and functional unit of mimicry supergene in female limited Batesian mimic Papilio butterflies

Abstract

It has long been suggested that dimorphic female-limited Batesian mimicry of two closely related Papilio butterflies, Papilio memnon and Papilio polytes, is controlled by supergenes. Whole-genome sequencing, genome-wide association studies and functional analyses have recently identified mimicry supergenes, including the doublesex (dsx) gene. Although supergenes of both the species are composed of highly divergent regions between mimetic and non-mimetic alleles and are located at the same chromosomal locus, they show critical differences in genomic architecture, particularly with or without an inversion: P. polytes has an inversion, but P. memnon does not. This review introduces and compares the detailed genomic structure of mimicry supergenes in two Papilio species, including gene composition, repetitive sequence composition, breakpoint/boundary site structure, chromosomal inversion and linkage disequilibrium. Expression patterns and functional analyses of the respective genes within or flanking the supergene suggest that dsx and other genes are involved in mimetic traits. In addition, structural comparison of the corresponding region for the mimicry supergene among further Papilio species suggests three scenarios for the evolution of the mimicry supergene between the two Papilio species. The structural features revealed in the Papilio mimicry supergene provide insight into the formation, maintenance and evolution of supergenes. This article is part of the theme issue 'Genomic architecture of supergenes: causes and evolutionary consequences'.

Keywords: Papilio butterflies; chromosomal inversion; female-limited polymorphic mimicry; linkage disequilibrium; supergene; transposon.

Figure 1.

(a) Wing patterns of adult males and non-mimetic and mimetic females of Papilio polytes and Papilio memnon. Mimicry is regulated by the H locus for P. polytes and A locus for P. memnon, with the mimetic allele (H or A) dominant over the non-mimetic allele (h or a). (b) Comparison of the detailed structure of mimicry highly divergent regions (HDRs) in P. polytes and P. memnon [8,9]. The direction of the dsx-HDR is reversed between h and H of P. polytes, but is the same between a and A of P. memnon. Putative breakpoints of the HDRs in P. polytes or boundary sites in P. memnon are indicated using red or green dotted lines, respectively. Graphical overview of the homology between heterozygous regions is also shown. Exon regions are shown in blue. (c) Enlarged view of the 20 kb region around the left breakpoint in P. polytes. U3X is transcribed only from H. The two dotted boxes with red shaded lines indicate the read-through transcripts of prospero. Some RNA-sequencing reads were mapped on H but not on h in this region [8]. (d) An enlarged view of the 200 bp region near the left breakpoint/boundary site in P. polytes and P. memnon. UXT crosses the breakpoint/boundary site at its 5'-untranslated region (UTR) in P. polytes and P. memnon (blue and red boxes, respectively); therefore, the sequences between the transcriptional start point and 95 bp region upstream of a translational start are very different [9].

Figure 2.

(a–c) Linkage disequilibrium (LD) heatmaps around the mimicry HDRs in P. polytes (a), P. memnon (b) and P. rumanzovia (c). LD across the mimicry HDR and the flanking 50 kb region. Standard colour scheme: D < 1, LOD < 2 (white); D'=1, LOD < 2 (blue); D' ≤ 1, LOD ≥ 2 (pink and red). Re-analyses were conducted using publicly available genome sequencing data [8,9,24,25,27]. Platanus-allee (v. 2.3.2) [27] was used to perform whole genome assembly using individuals of dsx genotypes Hh in P. polytes (accession number: SAMD00131361) and Aa in P. memnon (accession number: SAMD00074273). Using these as the reference genome, mapping was performed using six individuals (accession numbers: SRR1118145, SRR1118150, SRR1118152, SRR1111718, SRR1112070 and SRR1112619) for P. polytes, and 11 individuals (accession numbers: SAMD00074266–SAMD00074276) for P. memnon. Reads were mapped using BWA (v. 0.7.17-r1188) [28], single nucleotide variants (SNVs) were detected using GATK (v. 4.2.2.0) [29], and the file format of the SNV detection result was converted for Haploview [30] using PLINK (v. 1.90b6.24) [31]. The number of SNVs was set to 2000. In P. rumanzovia, LD was calculated by mapping 46 samples (accession numbers: SAMN13265081–SAMN13265107 and SAMN13265109–SAMN13265127) to the reference genome of P. memnon. (d) Percentages of the various types of repetitive sequences in the whole genome and within the mimicry HDR. The proportion of repetitive sequences was calculated using the same whole genomes, as in (a–c), with RepeatMasker (v4.0.9; www.repeatmasker.org/).

Figure 3.

Summary of expression and function of genes within and flanking the mimicry HDR in P. polytes. The expression levels of dsx-H, dsx-h, UXT-H, UXT-h, U3X, prospero and sir2 in mimetic and non-mimetic females and males of hindwings are summarized based on Komata et al. [26]. W indicates the wandering stage of the late last instar larvae; EP (P2) indicates the early pupa (2 day after pupation: P2); MP (P5) indicates the middle pupa (5 day after pupation: P5). In addition, the genetic functions and characteristics were shown. Nach-like was not included in the table because its expression in the wing has not been confirmed and its function is not known [9,26].

Figure 4.

(a) A sketch of the phylogeny and occurrence of female-limited mimicry polymorphism in the subgenus Menelaides of the genus Papilio. The cladogram is drawn based on [25]. Species in bold show female-limited mimicry polymorphism. The inversion in the mimicry HDR is present in P. polytes (red) and absent in P. memnon (green) and P. rumanzovia (green). Papilio xuhus is an outgroup. (b) Three possible genealogies under female-limited mimicry polymorphism in the subgenus Menelaides. The causative locus for polymorphism in P. aegeus is not clear, and the evolutionary scenarios among P. polytes, P. memnon, and P. rumanzovia are shown. (c) Model diagram of the evolutionary process in the mimicry HDR of P. polytes, P. memnon and P. rumanzovia. There are three processes depending on when the inversion occurs. In the case of independent evolution, the mimetic allele (H/A) first arises independently with a chromosome inversion or without an inversion (by transposable element or In-Del) at chromosome 25. The differentiation of the mimetic allele by a chromosome inversion is in P. polytes, while the differentiation without an inversion is in P. memnon and P. rumanzovia. In terms of the ancestral polymorphism-1, the mimetic alleles initially differentiate without an inversion in the common ancestor of the three species, followed by a chromosome inversion, resulting in the P. polytes mimetic allele. Furthermore, in ancestral polymorphism-2, the first inversion in the common ancestor, followed by re-inversion, is considered to be the mimetic allele of P. memnon. The left breakpoint/boundary site of the HDR is preserved, as shown in §2 and figure 2. In P. rumanzovia, the right boundary site is shifted to the left, resulting in a smaller HDR than in P. memnon. Red arrows indicate independent evolution, blue arrows indicate ancestral polymorphism-1, and red arrows indicate ancestral polymorphism-2, as discussed in figure 4b.